Probiotic Bifidobacterium strains

ABSTRACT

Probiotic  Bifidobacterium  strain AH1205 or mutants or variants thereof are immunomodulatory following oral consumption and are useful in the prophylaxis and/or treatment of inflammatory activity such as undesirable gastrointestinal inflammatory activity for example inflammatory bowel disease.

This is a national stage of PCT/IE08/000,034 filed Mar. 28, 2008 andpublished in English, claiming benefit of U.S. provisional applicationNo. 60/907,312, filed Mar. 28, 2007, hereby incorporated by reference.

The invention relates to a Bifidobacterium strain and its use as aprobiotic bacteria in particular as an immunomodulatory biotherapeuticagent.

The defense mechanisms to protect the human gastrointestinal tract fromcolonization by intestinal bacteria are highly complex and involve bothimmunological and non-immunological aspects (1). Innate defensemechanisms include the low pH of the stomach, bile salts, peristalsis,mucin layers and anti-microbial compounds such as lysozyme (2).Immunological mechanisms include specialized lymphoid aggregates,underlying M cells, called peyers patches which are distributedthroughout the small intestine and colon (3). Luminal antigens presentedat these sites result in stimulation of appropriate T and B cell subsetswith establishment of cytokine networks and secretion of antibodies intothe gastrointestinal tract (4). In addition, antigen presentation mayoccur via epithelial cells to intraepithelial lymphocytes and to theunderlying lamina propria immune cells (5). Therefore, the host investssubstantially in immunological defense of the gastrointestinal tract.However, as the gastrointestinal mucosa is the largest surface at whichthe host interacts with the external environment, specific controlmechanisms must be in place to regulate immune responsiveness to the 100tons of food which is handled by the gastrointestinal tract over anaverage lifetime. Furthermore, the gut is colonized by over 500 speciesof bacteria numbering 10¹¹-10¹²/g in the colon. Thus, these controlmechanisms must be capable of distinguishing non-pathogenic adherentbacteria from invasive pathogens, which would cause significant damageto the host. In fact, the intestinal flora contributes to defense of thehost by competing with newly ingested potentially pathogenicmicro-organisms.

Bacteria present in the human gastrointestinal tract can promoteinflammation. Aberrant immune responses to the indigenous microflorahave been implicated in certain disease states, such as inflammatorybowel disease. Antigens associated with the normal flora usually lead toimmunological tolerance and failure to achieve this tolerance is a majormechanism of mucosal inflammation (6). Evidence for this breakdown intolerance includes an increase in antibody levels directed against thegut flora in patients with inflammatory bowel disease (IBD).

The present invention is directed towards a Bifidobacterium strain whichhas been shown to have immunomodulatory effects, by modulating cytokinelevels or by antagonizing and excluding pro-inflammatory micro-organismsfrom the gastrointestinal tract.

STATEMENTS OF INVENTION

According to the invention there is provided Bifidobacterium strainAH1205 (NCIMB41387) or mutants or variants thereof.

The mutant may be a genetically modified mutant. The variant may be anaturally occurring variant of Bifidobacterium.

The strain may be a probiotic. It may be in the form of a biologicallypure culture.

The invention also provides an isolated strain of Bifidobacterium NCIMB41387

In one embodiment of the invention Bifidobacterium strains are in theform of viable cells. Alternatively Bifidobacterium strains are in theform of non-viable cells.

In one embodiment of the invention the Bifidobacterium strains areisolated from infant faeces, the Bifidobacterium strains beingsignificantly immunomodulatory following oral consumption in humans.

The invention also provides a formulation which comprises theBifidobacterium strain of the invention.

In one embodiment of the invention the formulation includes anotherprobiotic material.

In one embodiment of the invention the formulation includes a prebioticmaterial.

Preferably the formulation includes an ingestable carrier. Theingestable carrier may be a pharmaceutically acceptable carrier such asa capsule, tablet or powder. Preferably the ingestable carrier is a foodproduct such as acidified milk, yoghurt, frozen yoghurt, milk powder,milk concentrate, cheese spreads, dressings or beverages.

In one embodiment of the invention the formulation of the inventionfurther comprises a protein and/or peptide, in particular proteinsand/or peptides that are rich in glutamine/glutamate, a lipid, acarbohydrate, a vitamin, mineral and/or trace element.

In one embodiment of the invention the Bifidobacterium strain is presentin the formulation at more than 10⁶ cfu per gram of delivery system.Preferably the formulation includes any one or more of an adjuvant, abacterial component, a drug entity or a biological compound.

In one embodiment of the invention the formulation is for immunisationand vaccination protocols.

The invention further provides a Bifidobacterium strain or a formulationof the invention for use as foodstuffs, as a medicament, for use in theprophylaxis and/or treatment of undesirable inflammatory activity, foruse in the prophylaxis and/or treatment of undesirable respiratoryinflammatory activity such as asthma, for use in the prophylaxis and/ortreatment of undesirable gastrointestinal inflammatory activity such asinflammatory bowel disease eg. Crohns disease or ulcerative colitis,irritable bowel syndrome, pouchitis, or post infection colitis, for usein the prophylaxis and/or treatment of gastrointestinal cancer(s), foruse in the prophylaxis and/or treatment of systemic disease such asrheumatoid arthritis, for use in the prophylaxis and/or treatment ofautoimmune disorders due to undesirable inflammatory activity, for usein the prophylaxis and/or treatment of cancer due to undesirableinflammatory activity, for use in the prophylaxis of cancer, for use inthe prophylaxis and/or treatment of diarrhoeal disease due toundesirable inflammatory activity, such as Clostridium difficileassociated diarrhoea, Rotavirus associated diarrhoea or post infectivediarrhoea, for use in the prophylaxis and/or treatment of diarrhoealdisease due to an infectious agent, such as E. coli.

The invention also provides a Bifidobacterium strain or a formulation ofthe invention for use in the preparation of an anti-inflammatorybiotherapeutic agent for the prophylaxis and/or treatment of undesirableinflammatory activity or for use in the preparation of anti-inflammatorybiotherapeutic agents for the prophylaxis and/or treatment ofundesirable inflammatory activity.

In one embodiment of the invention the strain of the invention act byantagonising and excluding proinflammatory micro-organisms from thegastrointestinal tract.

The invention also provides a Bifidobacterium strain or a formulation ofthe invention for use in the preparation of anti-inflammatorybiotherapeutic agents for reducing the levels of pro-inflammatorycytokines.

The invention further provides a Bifidobacterium strain for use in thepreparation of anti-inflammatory biotherapeutic agents for modifying thelevels of IL-10.

The invention also provides for the use of a Bifidobacterium strain as aanti-infective probiotic due to their ability to antagonise the growthof pathogenic species.

We have found that particular strains of Bifidobacterium elicitimmunomodulatory effects in vitro.

The invention is therefore of major potential therapeutic value in theprophylaxis or treatment of dysregulated immune responses, such asundesirable inflammatory reactions for example asthma.

Bifidobacterium are commensal microorganisms. They have been isolatedfrom the microbial flora within the human gastrointestinal tract. Theimmune system within the gastrointestinal tract cannot have a pronouncedreaction to members of this flora, as the resulting inflammatoryactivity would also destroy host cells and tissue function. Therefore,some mechanism(s) exist whereby the immune system can recognizecommensal non-pathogenic members of the gastrointestinal flora as beingdifferent to pathogenic organisms. This ensures that damage to hosttissues is restricted and a defensive barrier is still maintained.

A deposit of Bifidobacterium longum strain AH1205 was made at the NCIMBon May 11, 2006 and accorded the accession number NCIMB 41387.

The Bifidobacterium longum may be a genetically modified mutant or itmay be a naturally occurring variant thereof.

Preferably the Bifidobacterium longum is in the form of viable cells.

Alternatively the Bifidobacterium longum may be in the form ofnon-viable cells.

It will be appreciated that the specific Bifidobacterium strain of theinvention may be administered to animals (including humans) in an orallyingestible form in a conventional preparation such as capsules,microcapsules, tablets, granules, powder, troches, pills, suppositories,suspensions and syrups. Suitable formulations may be prepared by methodscommonly employed using conventional organic and inorganic additives.The amount of active ingredient in the medical composition may be at alevel that will exercise the desired therapeutic effect.

The formulation may also include a bacterial component, a drug entity ora biological compound.

In addition a vaccine comprising the strains of the invention may beprepared using any suitable known method and may include apharmaceutically acceptable carrier or adjuvant.

Throughout the specification the terms mutant, variant and geneticallymodified mutant include a strain of Bifidobacteria whose genetic and/orphenotypic properties are altered compared to the parent strain.Naturally occurring variant of Bifidobacterium longum includes thespontaneous alterations of targeted properties selectively isolated.Deliberate alteration of parent strain properties is accomplished byconventional (in vitro) genetic manipulation technologies, such as genedisruption, conjugative transfer, etc. Genetic modification includesintroduction of exogenous and/or endogenous DNA sequences into thegenome of a Bifidobacteria strain, for example by insertion into thegenome of the bacterial strain by vectors, including plasmid DNA, orbacteriophages.

Natural or induced mutations include at least single base alterationssuch as deletion, insertion, transversion or other DNA modificationswhich may result in alteration of the amino acid sequence encoded by theDNA sequence.

The terms mutant, variant and genetically modified mutant also include astrain of Bifidobacteria that has undergone genetic alterations thataccumulate in a genome at a rate which is consistent in nature for allmicro-organisms and/or genetic alterations which occur throughspontaneous mutation and/or acquisition of genes and/or loss of geneswhich is not achieved by deliberate (in vitro) manipulation of thegenome but is achieved through the natural selection of variants and/ormutants that provide a selective advantage to support the survival ofthe bacterium when exposed to environmental pressures such asantibiotics. A mutant can be created by the deliberate (in vitro)insertion of specific genes into the genome which do not fundamentallyalter the biochemical functionality of the organism but whose productscan be used for identification or selection of the bacterium, forexample antibiotic resistance.

A person skilled in the art would appreciate that mutant or variantstrains of Bifidobacteria can be identified by DNA sequence homologyanalysis with the parent strain. Strains of Bifidobacteria having aclose sequence identity with the parent strain are considered to bemutant or variant strains. A Bifidobacteria strain with a sequenceidentity (homology) of 96% or more, such as 97% or more, or 98% or more,or 99% or more with the parent DNA sequence may be considered to be amutant or variant. Sequence homology may be determined using on-linehomology algorithm “BLAST” program, publicly available athttp://www.ncbi.nlm.nih,gov/BLAST/.

Mutants of the parent strain also include derived Bifidobacteria strainshaving at least 85% sequence homology, such as at least 90% sequencehomology, or at least 95% sequence homology to the 16s-23s intergenicspacer polynucleotide sequence of the parent strain. These mutants mayfurther comprise DNA mutations in other DNA sequences in the bacterialgenome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a BOX PCR (BIOANALYZER) barcode profile for B. longum AH1205.Base pair sizes were determined using the AGILENT 2100 software.

FIG. 2 is a graph illustrating the faecal recovery of B. longum AH1205over an 8 day feeding period which demonstrates the ability of AH1205 totransit the murine gastrointestinal tract.

FIG. 3 is a bar graph showing the effect of B. longum AH1205 on IL-10cytokine production by human PBMCs. Results are expressed as mean+/−SE(n=6).

FIGS. 4 A and B are graphs showing the effect of probiotic bacterialstrain AH1205 (A) and placebo (B) on total cell numbers inbronchoalveolar lavage fluid following ovalbumin (OVA) challenge insensitised animals (n=10/group, *=p<0.05 compared to OVA challengealone).

FIGS. 5 A and B are graphs showing the effect of probiotic strain AH1205treatment (A) and placebo (B) on airway responsiveness to methacholine,as assessed by changes in enhanced pause (Penh) in ovalbumin(OVA)—sensitised mice 24 hours after intranaval challenge with OVA orsaline. Each data paint represents the mean±SEM (n=10/groups * p=<0.05compared to OVA alone).

FIG. 6 A to E are graphs showing IL-10(A), IFNγ(B), TNF(C), IL-6(D) andCCL2(E) cytokine levels in bronchoalveolar lavage (BAL) fluid fromovalbumin (OVA)—sensitised mice. Each column represents the mean±SEM(n=10, * p<0.05 compared to OVA challenged, saline treated control).

FIG. 7 is a graph illustrating that CD4⁺ CD25⁺ Cells from AH1205 fedmice significantly reduced responder CD4 T cell proliferation (n=7).

FIG. 8 is a graph showing the percentage of Payer's patch cells in theCD4+population that are also CD25+, as assessed by flow cytometry.

FIGS. 9 A and B are graphs showing the percentage of CD4/CD25+cellsexpressing the transcription factor Foxp3 is significantly upregulatedin germ free mice consuming AH1205. (A)=spleen cells, (B)=MLNC cells(n=4/group for spleen assay, n=2/3 for MLNC assay).

FIG. 10 is a graph showing that the level of cytokines IL-6, MCP-1 andIFN-γ secreted by CD3/CD28 stimulated MLNC cultures was reduced whengerm—free mice consumed Bifidobacterium longum AH1205. Results areexpressed as the mean per group+/−standard error. (n=4/group).

FIG. 11 is a graph showing that the level of cytokines IL-6 and TNF-αsecreted by CD3/CD28 stimulated splenocyte cultures was reduced whengerm—free mice consumed Bifidobacterium longum AH1205. Results areexpressed as the mean per group+/−standard error (n=4/group). and

FIG. 12 is a graph illustrating the stability of probiotic strain AH1205over 3 months compared to Lactobacillus GG.

DETAILED DESCRIPTION

We have found that Bifidobacterium longum strain AH1205 is not only acidand bile tolerant and transits the gastrointestinal tracts but also,surprisingly has immunomodulatory effects, by modulating cytokine levelsor by antagonising and excluding pro-inflammatory or immunomodulatorymicro-organisms from the gastrointestinal tract.

The general use of probiotic bacteria is in the form of viable cells.However, it can also be extended to non-viable cells such as killedcultures or compositions containing beneficial factors expressed by theprobiotic bacteria. This could include thermally killed micro-organismsor micro-organisms killed by exposure to altered pH or subjection topressure. With non-viable cells product preparation is simpler, cellsmay be incorporated easily into pharmaceuticals and storage requirementsare much less limited than viable cells. Lactobacillus casei YIT 9018offers an example of the effective use of heat killed cells as a methodfor the treatment and/or prevention of tumour growth as described inU.S. Pat. No. 4,347,240.

It is unknown whether intact bacteria are required to exert animmunomodulatory effect or if individual active components of theinvention can be utilized alone. Proinflammatory components of certainbacterial strains have been identified. The proinflammatory effects ofgram-negative bacteria are mediated by lipopolysaccharide (LPS). LPSalone induces a proinflammatory network, partially due to LPS binding tothe CD14 receptor on monocytes. It is assumed that components ofprobiotic bacteria possess immunomodulatory activity, due to the effectsof the whole cell. Upon isolation of these components, pharmaceuticalgrade manipulation is anticipated.

IL-10 is produced by T cells, B cells, monocytes and macrophages. Thiscytokine augments the proliferation and differentiation of B cells intoantibody secreting cells. IL-10 exhibits mostly anti-inflammatoryactivities. It up-regulates IL-1RA expression by monocytes andsuppresses the majority of monocyte inflammatory activities. IL-10inhibits monocyte production of cytokines, reactive oxygen and nitrogenintermediates, MHC class II expression, parasite killing and IL-10production via a feed back mechanism (7). This cytokine has also beenshown to block monocyte production of intestinal collagenase and type IVcollagenase by interfering with a PGE₂-cAMP dependant pathway andtherefore may be an important regulator of the connective tissuedestruction seen in chronic inflammatory diseases.

The host response to infection is characterised by innate and acquiredcellular and humoral immune reactions, designed to limit spread of theoffending organism and to restore organ homeostasis. However, to limitthe aggressiveness of collateral damage to host tissues, a range ofregulatory constraints may be activated. Regulatory T cells (Tregs)serve one such mechanism. These are derived from the thymus but may alsobe induced in peripheral organs, including the gut mucosa. Deliberateadministration of Treg cells suppresses inflammatory disease in a widerange of murine models including experimental autoimmuneencephalomyelitis, inflammatory bowel disease, bacterial-inducedcolitis, collagen-induced arthritis, type I diabetes, airway osinophilicinflammation, graft-vs-host disease and organ transplantation. Theforkhead transcription factor Foxp3 (forkhead box P3) is selectivelyexpressed in Treg cells, is required for Treg development and function,and is sufficient to induce a Treg phenotype in conventional CD4 cells(19). Mutations in Foxp3 cause severe, multi-organ autoimmunity in bothhuman and mouse. We have described a Bifidobacterium strain thatgenerates CD25 positive/Foxp3 positive T regulatory cells in vivo.

The invention will be more clearly understood from the followingexamples.

EXAMPLE 1 Characterisation of Bacteria Isolated from Infant Faeces

Demonstration of Probiotic Traits

Isolation of Probiotic Bacteria

Fresh faeces was obtained from a 3 day old male breast fed infant andserially dilutions were plated on TPY (trypticase, peptone and yeastextract) and MRS (deMann, Rogosa and Sharpe) media supplemented with0.05% cysteine and mupirocin. Plates were incubated in anaerobic jars(BBL, Oxoid) using CO₂ generating kits (Anaerocult A, Merck) for 2-5days at 37° C. Gram positive, catalase negative rod-shaped orbifurcated/pleomorphic bacteria isolates were streaked for purity on tocomplex non-selective media (MRS and TPY). Isolates were routinelycultivated in MRS or TPY medium unless otherwise stated at 37° C. underanaerobic conditions. Presumptive Bifidobacterium were stocked in 40%glycerol and stored at −20° C. and −80° C.

Following isolation of a pure bifidobacteria strain, assigned thedesignation AH1205, microbiological characteristics were assessed andare summarized in Table 1 below. AH1205 is a gram positive, catalasenegative pleomorphic shaped bacterium which is Fructose-6-PhoshatePhosphoketolase positive confirming its identity as a bifidobacterium.Using minimal media in which a single carbon source was inserted, AH1205was able to grow on all carbon sources tested (Glucose, Lactose, Ribose,Arabinose, Galactose, Raffinose, Fructose, Malt Extract, Mannose,Maltose, Sucrose).

TABLE 1 Physiochemical characteristics of B. longum AH1205 B. longumAH1205 Strain Characteristics Gram Stain + Catalase − Motility −F6PPK* + Milk coagulation + 45° C. anaerobic culture − 45° C. aerobicculture − CHO Fermentation: Glucose + Lactose + Ribose + Arabinose +Galactose + Raffinose + Fructose + Malt Extract + Mannose + Maltose +Sucrose + *signifies Fructose-6-Phoshate Phosphoketolase AssaySpecies Identification

16s intergenic spacer (IGS) sequencing was performed to identify thespecies of bifidobacteria isolated. Briefly, DNA was isolated fromAH1205 using 100 μl of Extraction Solution and 25 μl of TissuePreparation solution (Sigma, XNAT2 Kit). The samples were incubated for5 minutes at 95° C. and then 100 μl of Neutralization Solution (XNAT2kit) was added. Genomic DNA solution was quantified using a NANODROPspectrophotometer and stored at 4° C. PCR was performed using the IGSprimers, IGS L: 5′-GCTGGATCACCTCCTTTC-3′ (SEQ ID NO. 3) which is basedon SEQ ID NO. 1 and IGS R: 5′-CTGGTGCCAAGGCATCCA-3′ (SEQ ID NO. 4) whichis based on SEQ ID NO. 2. The cycling conditions were 94° C. for 3 min(1 cycle), 94° C. for 30 sec, 53° C. for 30 sec, 72° C. for 30 sec (28cycles). The PCR reaction contained 4 μl (50 ng) of DNA, PCR mix (XNAT2kit), 0.4 μM IGS L and R primer (MWG Biotech, Germany). The PCRreactions were performed on an EPPENDORF THERMOCYCLER. The PCR products(10 μl) were ran alongside a molecular weight marker (100 bp Ladder,Roche) on a 2% agarose EtBr stained gel in TAE, to determine the IGSprofile. PCR products of Bifidobacterium (single band) were purifiedusing the PROMEGA WIZARD PCR purification kit. The purified PCR productswere sequenced using the primer sequences (above) for the intergenicspacer region. Sequence data was then searched against the NCBInucleotide database to determine the identity of the strain bynucleotide homology. The resultant DNA sequence data was subjected tothe NCBI standard nucleotide-to-nucleotide homology BLAST search engine(http://www.ncbi.nlm.nih.gov/BLAST/). The nearest match to the sequencewas identified and then the sequences were aligned for comparison usingDNASTAR MegAlign software. The sequences (SEQ ID NO. 1 [IGS forwardsequence] and SEQ ID NO. 2 [IGS reverse sequence]) obtained can beviewed in the sequence listing. Searching the NCIMB database revealedthat AH1205 has a unique IGS (SEQ ID NO. 1 [forward sequence] and SEQ IDNO. 2 [reverse sequence]) sequence with its closest sequence homology toa Bifidobacterium longum.

In order to develop a barcode PCR profile for AH1205, PCR was performedusing BOX primers (8). The cycling conditions were 94° C. for 7 min (1cycle); 94° C. for 1 minute, 65° C. for 8 minutes, (30 cycles) and 65°C. for 16 minutes. The PCR reaction contained 50 ng of DNA, PCR mix(XNAT2 kit) and 0.3 μM BOXA1R primer (5′-CTACGGCAAGGCGACGCTGACG-3′) (SEQID NO. 5) (MWG Biotech, Germany). The PCR reactions were performed on anEPPENDORF THERMOCYCLER. The PCR products (1 μl) were ran alongside amolecular weight marker (DNA 7500 ladder, Agilent, Germany) using theDNA 7500 LABCHIP on the AGILENT 2100 BIOANALYZER (Agilent, Germany). Thebarcode (PCR product profile) was determined using the AGILENTBIOANALYZER software where peak number (PCR products) and size wereidentified (FIG. 1).

Antibiotic Sensitivity Profiles

Antibiotic sensitivity profiles of the B. longum strain was determinedusing the ‘disc susceptibility’ assay. Cultures were grown up in theappropriate broth medium for 24-48 h spread-plated (100 μl) onto agarmedia and discs containing known concentrations of the antibiotics wereplaced onto the agar. Strains were examined for antibiotic sensitivityafter 1-2 days incubation at 37° C. under anaerobic conditions. Strainswere considered sensitive if zones of inhibition of 1 mm or greater wereseen. The minimum inhibitory concentration (MIC) for each antibiotic wasindependently assessed. The MIC for clindamycin, vancomycin andmetronidazole were 0.032, 0.75 and >256 respectively.

Intestinal Transit

To determine whether Bifidobacterium longum could survive at low pHvalues equivalent to those found in the stomach, bacterial cells wereharvested from fresh overnight cultures, washed twice in phosphatebuffer (pH 6.5) and resuspended in TPY broth adjusted to pH 2.5 (with 1MHCl). Cells were incubated at 37° C. and survival measured at intervalsof 5, 30, 60 and 120 minutes using the plate count method. AH1205survived well for 5 minutes at pH 2.5 while no viable cells wererecovered after 30 minutes.

Upon exiting the stomach, putative probiotics are exposed to bile saltsin the small intestine. In order to determine the ability of B. longumto survive exposure to bile, cultures were streaked on TPY agar platessupplemented with 0.3% (w/v), 0.5%, 1%, 2%, 5%, 7.5% or 10% porcinebile. B. longum AH1205 growth was observed on plates containing up to0.5% bile.

In a murine model, the ability of B. longum AH1205 to transit thegastrointestinal tract was assessed. Mice consumed 1×10⁹ AH1205 dailyand faecal pellets were examined for the presence of the fedmicro-organism. Detection of AH1205 was facilitated by isolating aspontaneous rifampicin resistant variant of thebifidobacteria—incorporation of rifampicin in the TPY plates used toassess transit ensured that only the fed rifampicin resistantbifiobacteria was cultured. Faecal samples were collected daily and B.longum transit through the gastrointestinal tract was confirmed (FIG.2).

Anti-Microbial Activity

The indicator pathogenic micro-organisms used in this study werepropagated in the following medium under the following growthconditions: Salmonella typhimurium (37° C., aerobic) in Tryptone Soyabroth/agar supplemented with 0.6% yeast extract (TSAYE, Oxoid),Campylobacter jejuni (37° C., anaerobic) and E. coli O157:H7 (37° C.,anaerobic) on Blood agar medium, Clostridium difficile (37° C.,anaerobic) in reinforced Clostridial medium (RCM, Oxoid). All strainswere inoculated into fresh growth medium and grown overnight beforebeing used in experiments.

Antimicrobial activity was detected using the deferred method (9).Briefly, B. longum AH1205 was incubated for 36-48 h. Ten-fold serialdilutions were spread-plated (100 μl) onto TPY agar medium. Afterovernight incubation, plates with distinct colonies were overlayed withthe indicator bacterium. The indicator lawn was prepared by inoculatinga molten overlay with 2% (v/v) of an overnight indicator culture whichwas poured over the surface of the inoculated TPY plates. The plateswere re-incubated overnight under conditions suitable for growth of theindicator bacterium. Indicator cultures with inhibition zones greaterthan 1 mm in radius were considered sensitive to the test bacterium. B.longum AH1205 inhibited the growth of all pathogenic organisms tested,with zones of clearing measuring 8.67, >80, 4.33 and 11.67 mm forSalmonella typhimurium, Campylobacter jejuni, E. coli O157:H7 andClostridium difficile respectively.

EXAMPLE 2 Cytokine Production by PBMCs in Response to B. Longum

Peripheral blood mononuclear cells (PBMCs) were isolated from healthydonors by density gradient centrifugation. PBMCs were stimulated withthe probiotic bacterial strain for a 72 hour period at 37° C. At thistime culture supernatants were collected, centrifuged, aliquoted andstored at −70° C. until being assessed for IL-10 levels using cytometricbead arrays (BD BioSciences). AH1205 induced significant secretion ofIL-10 by human PBMCs suggesting this probiotic strain would induce aanti-inflammatory response in vivo (FIG. 3).

EXAMPLE 3 B. Longum AH1205 Attenuates Respiratory Disease in a MurineModel of Asthma

This study investigated whether the probiotic bacteria Bifidobacteriumlongum AH1205, suppress allergic responses in an ovalbumin (OVA)sensitized mouse model of allergic airway inflammation. Briefly, adultmale BALB/c mice were sensitized by i.p. injection of OVA day 0 and day6. On days 12 and 14, mice were challenged intranasally with OVA.Twenty-four hours after the last challenge (day 15), mice were subjectedto measurements of airway responsiveness followed by BAL procedure.OVA/alum-sensitized, saline-challenged mice served as controls. Animalsreceived probiotic or placebo throughout the trial. Airway inflammation(cytokine and cell counts) was assessed by inflammatory cell counts inbronchoalveolar lavage (BAL) fluid. Airway responsiveness was alsomeasured using the Buxco whole-body plethysmograph. Splenocytes werealso isolated from OVA sensitized mice and were incubated in thepresence of anti-CD3 and anti-CD28 antibodies after which cytokinelevels were measured in the supernatants by flow cytometry.

B. longum AH1205 caused no significant reduction in cells recovered fromBAL fluid following OVA challenge, when compared to broth fed animals(FIG. 4). Airway responsiveness was measured and challenge of sensitizedmice with OVA resulted in an enhancement of AHR to methacholine whencompared with saline-challenged mice. AH1205 did not modulate thisenhanced airway responsiveness to methacholine, as assessed by changesin enhanced pause (FIG. 5).

BAL cytokine levels were measured by cytometric bead array anddemonstrated that animals fed AH1205 had significantly reduced TNF-αlevels compared to OVA control (FIG. 6C). No significant differenceswere noted for IL-10, IFN-γ, IL-6 and CCL2 levels. (FIG. 6)

EXAMPLE 4 Treg Effector Model

This study investigated the effect of probiotic consumption onregulatory T cell number and activity in healthy mice. BALB/c mice(10/group) were fed Bifidobacterium longum AH1205 or placebo for threeweeks. Following probiotic/placebo consumption, CD4+CD25+ T-regulatorycells were isolated and their in vitro suppressive activity wasdetermined by measuring proliferation of anti-CD3/CD28 stimulatedCFSE-labelled CD4+ responder T cells using flow cytometry. CD4+responder T cells were co-incubated with CD4+CD25− T cells as a control.The percentage of CD4+CD25+ cells (Regulatory T cells) in murinesplenocytes that are also FoxP3 positive was determined in the spleensof probiotic or placebo-fed mice.

The % of CD4+ cells that proliferated when co-incubated with CD4+CD25+cells from the probiotic/placebo fed mice was compared to the % of CD4+cells that proliferated when co-incubated with CD4+CD25− cells from thesame trial mouse. In each case, T cell proliferation was less incultures containing CD4+CD25+ cells compared in cultures containing CD4cells alone and depleted of the CD25+ cells (FIG. 7).

The % of cells in the CD4+ population that were also CD25+ wasdetermined (FIG. 8). The Bifidobacterium longum AH1205 fed group hadsignificantly more CD4+ T cells that were CD25+ (i.e. T-Regulatorycells) than their placebo-fed counterparts. This suggests that the % ofT-Regulatory cells within the CD4+ population was increasedsignificantly by feeding with AH1205.

The number of CD4+CD25+ FoxP3+ cells in the whole splenocyte populationsof probiotic or placebo-fed mice was also determined. The number ofCD4+CD25+ T-Regulatory cells expressing FoxP3 was unchanged in thespleens of probiotic fed mice relative to placebo or unfed mice.

EXAMPLE 5 Germ Free Model

Germ free mice were purchased at 6 weeks of age and maintained in thegerm-free unit at the biological services unit in UCC. Animals consumedthe probiotic strain Bifidobacterium longum AH1205 for 9 days orremained germ free. Induction of T regulatory cells was assessed by flowcytometry and cytokine levels were quantified by CBA.

AH1205 transit was assessed by measuring bifidobacterial counts onselective agar over the course of the study. AH1205 did not transit thegut of germ free mice in measurable numbers even though approximately1×10⁹ organisms were administered daily. The results suggests thatadditional microbial or host factors are required for bifidobacterialsurvival within the gut.

While AH1205 did not transit the gut in detectable numbers, the bacteriadid interact with the host immune system. The numbers of CD4+CD25+Foxp3+ cells in the mesenteric lymph node and spleen of AH1205 fedgerm-free animals was significantly increased following 9 days offeeding (FIG. 9). Total CD3/CD4 or CD3/CD8 counts remained unaltered.

Isolated mesenteric lymph node cells (MLNC) and splenocytes werestimulated in vitro with anti-CD3/CD28 antibodies or LPS or remainedun-stimulated as negative controls. MLNC secretion of IL-6 and IFN-γ,following CD3/CD28 stimulation, was substantially decreased in culturesupernatants while MCP-1 levels were significantly suppressed when micewere pre-fed AH1205 (FIG. 10). IL-10 levels remained similar between thegroups. Splenocyte release of IL-6 and TNF-α was substantially, but notsignificantly, decreased when pre-fed AH1205 (FIG. 11). No significantdifferences were noted for the un-stimulated or LPS stimulated culturesbut overall we observed less pro-inflammatory cytokine production fromthe Bifidobacteria AH1205-fed animals.

EXAMPLE 6 Stability Results

The stability of the probiotic strain AH1205, varied over 3 months at30° C. (FIG. 12)

Lactobacillus GG was a poor performer over the test period with a 2 logdrop over the 3 month period whereas strain AH 1205 declined inviability by up to approximately 1 log over the same test period.

Immunomodulation

The human immune system plays a significant role in the aetiology andpathology of a vast range of human diseases. Hyper and hypo-immuneresponsiveness results in, or is a component of, the majority of diseasestates. One family of biological entities, termed cytokines, areparticularly important to the control of immune processes. Pertubancesof these delicate cytokine networks are being increasingly associatedwith many diseases. These diseases include but are not limited toinflammatory disorders, immunodeficiency, inflammatory bowel disease,irritable bowel syndrome, cancer (particularly those of thegastrointestinal and immune systems), diarrhoeal disease, antibioticassociated diarrhoea, paediatric diarrhoea, appendicitis, autoimmunedisorders, multiple sclerosis, Alzheimer's disease, rheumatoidarthritis, coeliac disease, diabetes mellitus, organ transplantation,bacterial infections, viral infections, fungal infections, periodontaldisease, urogenital disease, sexually transmitted disease, HIVinfection, HIV replication, HIV associated diarrhoea, surgicalassociated trauma, surgical-induced metastatic disease, sepsis, weightloss, anorexia, fever control, cachexia, wound healing, ulcers, gutbarrier function, allergy, asthma, respiratory disorders, circulatorydisorders, coronary heart disease, anaemia, disorders of the bloodcoagulation system, renal disease, disorders of the central nervoussystem, hepatic disease, ischaemia, nutritional disorders, osteoporosis,endocrine disorders, epidermal disorders, psoriasis and acne vulgaris.The effects on cytokine production are specific for each of theprobiotic strains examined. Thus specific probiotic strains may beselected for normalizing an exclusive cytokine imbalance particular fora specific disease type. Customization of disease specific therapies canbe accomplished using either a single strain of AH1205 or mutants orvariants thereof or a selection of these strains.

Immune Education

The enteric flora is important to the development and proper function ofthe intestinal immune system. In the absence of an enteric flora, theintestinal immune system is underdeveloped, as demonstrated in germ freeanimal models, and certain functional parameters are diminished, such asmacrophage phagocytic ability and immunoglobulin production (10). Theimportance of the gut flora in stimulating non-damaging immune responsesis becoming more evident. The increase in incidence and severity ofallergies in the western world has been linked with an increase inhygiene and sanitation, concomitant with a decrease in the number andrange of infectious challenges encountered by the host. This lack ofimmune stimulation may allow the host to react to non-pathogenic, butantigenic, agents resulting in allergy or autoimmunity. Deliberateconsumption of a series of non-pathogenic immunomodulatory bacteriawould provide the host with the necessary and appropriate educationalstimuli for proper development and control of immune function.

Inflammation

Inflammation is the term used to describe the local accumulation offluid, plasma proteins and white blood cells at a site that hassustained physical damage, infection or where there is an ongoing immuneresponse. Control of the inflammatory response is exerted on a number oflevels (11). The controlling factors include cytokines, hormones (e.g.hydrocortisone), prostaglandins, reactive intermediates andleukotrienes. Cytokines are low molecular weight biologically activeproteins that are involved in the generation and control ofimmunological and inflammatory responses, while also regulatingdevelopment, tissue repair and haematopoiesis. They provide a means ofcommunication between leukocytes themselves and also with other celltypes. Most cytokines are pleiotrophic and express multiple biologicallyoverlapping activities. Cytokine cascades and networks control theinflammatory response rather than the action of a particular cytokine ona particular cell type (12). Waning of the inflammatory response resultsin lower concentrations of the appropriate activating signals and otherinflammatory mediators leading to the cessation of the inflammatoryresponse. TNFα is a pivotal proinflammatory cytokine as it initiates acascade of cytokines and biological effects resulting in theinflammatory state. Therefore, agents which inhibit TNFα are currentlybeing used for the treatment of inflammatory diseases, e.g. INFLIXIMAB.

Pro-inflammatory cytokines are thought to play a major role in thepathogenesis of many inflammatory diseases, including inflammatory boweldisease (IBD). Current therapies for treating IBD are aimed at reducingthe levels of these pro-inflammatory cytokines, including IL-8 and TNFα.Such therapies may also play a significant role in the treatment ofsystemic inflammatory diseases such as rheumatoid arthritis.

The strains of the present invention may have potential application inthe treatment of a range of inflammatory diseases, particularly if usedin combination with other anti-inflammatory therapies, such asnon-steroid anti-inflammatory drugs (NSAIDs) or INFLIXIMAB.

Cytokines and Cancer

The production of multifunctional cytokines across a wide spectrum oftumour types suggests that significant inflammatory responses areongoing in patients with cancer. It is currently unclear what protectiveeffect this response has against the growth and development of tumourcells in vivo. However, these inflammatory responses could adverselyaffect the tumour-bearing host. Complex cytokine interactions areinvolved in the regulation of cytokine production and cell proliferationwithin tumour and normal tissues (13,14). It has long been recognizedthat weight loss (cachexia) is the single most common cause of death inpatients with cancer and initial malnutrition indicates a poorprognosis. For a tumour to grow and spread it must induce the formationof new blood vessels and degrade the extracellular matrix. Theinflammatory response may have significant roles to play in the abovemechanisms, thus contributing to the decline of the host and progressionof the tumour. Due to the anti-inflammatory properties ofBifidobacterium longum infantis these bacterial strains they may reducethe rate of malignant cell transformation. Furthermore, intestinalbacteria can produce, from dietary compounds, substances with genotoxic,carcinogenic and tumour-promoting activity and gut bacteria can activatepro-carcinogens to DNA reactive agents (15). In general, species ofBifidobacterium have low activities of xenobiotic metabolizing enzymescompared to other populations within the gut such as bacteroides,eubacteria and clostridia. Therefore, increasing the number ofBifidobacterium bacteria in the gut could beneficially modify the levelsof these enzymes.

Vaccine/Drug Delivery

The majority of pathogenic organisms gain entry via mucosal surfaces.Efficient vaccination of these sites protects against invasion by aparticular infectious agent. Oral vaccination strategies haveconcentrated, to date, on the use of attenuated live pathogenicorganisms or purified encapsulated antigens (16). Probiotic bacteria,engineered to produce antigens from an infectious agent, in vivo, mayprovide an attractive alternative as these bacteria are considered to besafe for human consumption (GRAS status).

Murine studies have demonstrated that consumption of probiotic bacteriaexpressing foreign antigens can elicit protective immune responses. Thegene encoding tetanus toxin fragment C (TTFC) was expressed inLactococcus lactis and mice were immunized via the oral route. Thissystem was able to induce antibody titers significantly high enough toprotect the mice from lethal toxin challenge. In addition to antigenpresentation, live bacterial vectors can produce bioactive compounds,such as immunostimulatory cytokines, in vivo. L. lactis secretingbioactive human IL-2 or IL-6 and TTFC induced 10-15 fold higher serumIgG titres in mice immunized intranasally (17). However, with thisparticular bacterial strain, the total IgA level was not increased bycoexpression with these cytokines. Other bacterial strains, such asStreptococcus gordonii, are also being examined for their usefulness asmucosal vaccines. Recombinant S. gordonii colonizing the murine oral andvaginal cavities induced both mucosal and systemic antibody responses toantigens expressed by this bacterial (18). Thus oral immunization usingprobiotic bacteria as vectors would not only protect the host frominfection, but may replace the immunological stimuli that the pathogenwould normally elicit thus contributing to the immunological educationof the host.

Prebiotics

The introduction of probiotic organisms is accomplished by the ingestionof the micro-organism in a suitable carrier. It would be advantageous toprovide a medium that would promote the growth of these probioticstrains in the large bowel. The addition of one or moreoligosaccharides, polysaccharides, or other prebiotics enhances thegrowth of lactic acid bacteria in the gastrointestinal tract. Prebioticsrefers to any non-viable food component that is specifically fermentedin the colon by indigenous bacteria thought to be of positive value,e.g. bifidobacteria, lactobacilli. Types of prebiotics may include thosethat contain fructose, xylose, soya, galactose, glucose and mannose. Thecombined administration of a probiotic strain with one or more prebioticcompounds may enhance the growth of the administered probiotic in vivoresulting in a more pronounced health benefit, and is termed synbiotic.

Other Active Ingredients

It will be appreciated that the probiotic strains may be administeredprophylactically or as a method of treatment either on its own or withother probiotic and/or prebiotic materials as described above. Inaddition, the bacteria may be used as part of a prophylactic ortreatment regime using other active materials such as those used fortreating inflammation or other disorders especially those with animmunological involvement. Such combinations may be administered in asingle formulation or as separate formulations administered at the sameor different times and using the same or different routes ofadministration.

The invention is not limited to the embodiments herein before describedwhich may be varied in detail.

REFERENCES

-   1. McCracken V. J. and Gaskins H. R. Probiotics and the immune    system. In: Probiotics a critical review, Tannock, G W (ed), Horizon    Scientific Press, UK. 1999, p. 85-113.-   2. Savage D. C. Interaction between the host and its microbes. In:    Microbial Ecology of the Gut, Clark and Bauchop (eds), Academic    Press, London. 1977, p. 277-310.-   3. Kagnoff M. F. Immunology of the intestinal tract. Gastroenterol.    1993; 105 (5): 1275-80.-   4. Lamm M. E. Interaction of antigens and antibodies at mucosal    surfaces. Ann. Rev. Microbiol. 1997; 51: 311-40.-   5. Raychaudhuri S., Rock K L. Fully mobilizing host defense:    building better vaccines. Nat biotechnol., 1998; 16: 1025-31.-   6. Stallmach A., Strober W, MacDonald T T, Lochs H, Zeitz M.    Induction and modulation of gastrointestinal inflammation. Immunol.    Today, 1998; 19 (10): 438-41.-   7. de Waal Malefyt R, Haanen J, Spits H, Roncarolo M G, to Velde A,    Figdor C, Johnson K, Kastelein R, Yssel H, de Vries J E. Interleukin    10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T    cell proliferation by diminishing the antigen-presenting capacity of    monocytes via downregulation of class II major histocompatibility    complex expression. J Exp Med 1991 Oct. 1; 174(4):915-24.-   8. Masco L, Huys G, Gevers D, Verbrugghen L, Swings J.    Identification of Bifidobacterium species using rep-PCR    fingerprinting. Syst Appl Microbiol. 2003 November; 26(4):557-63.    PMID: 14666984.-   9. Tagg, J R, Dajani, A S, Wannamaker, L W. Bacteriocins of Gram    positive bacteria. Bacteriol Rev. 1976; 40: 722-756.-   10. Crabbe P. A., H. Bazin, H. Eyssen, and J. F. Heremans. The    normal microbial flora as a major stimulus for proliferation of    plasma cells synthesizing IgA in the gut. The germ free intestinal    tract. Into. Arch. Allergy Appl Immunol, 1968; 34: 362-75.-   11. Henderson B., Poole, S and Wilson M. 1998. In “Bacteria-Cytokine    interactions in health and disease. Portland Press, 79-130.-   12. Arai K I, Lee F, Miyajima A, Miyatake S, Arai N, Yokota T.    Cytokines: coordinators of immune and inflammatory responses. Annu    Rev Biochem 1990; 59:783-836.-   13. McGee D W, Bamberg T, Vitkus S J, McGhee J R. A synergistic    relationship between TNF-alpha, IL-1 beta, and TGF-beta 1 on IL-6    secretion by the IEC-6 intestinal epithelial cell line. Immunology    1995 September; 86(1):6-11.-   14. Wu S, Meeker W A, Wiener J R, Berchuck A, Bast R C Jr, Boyer    C M. Transfection of ovarian cancer cells with tumour necrosis    factor alpha (TNF-alpha) antisense mRNA abolishes the proliferative    response to interleukin-1 (IL-1) but not TNF-alpha. Gynecol Oncol    1994 April; 53(1):59-63.-   15. Rowland I. R. Toxicology of the colon: role of the intestinal    microflora. In: Gibson G. R. (ed). Human colonic bacteria: role in    nutrition, physiology and pathology, 1995, pp 155-174. Boca Raton    CRC Press.-   16. Walker, R. I. New strategies for using mucosal vaccination to    achieve more effective immunization. Vaccine, 1994; 12: 387-400.-   17. Steidler L., K. Robinson, L. Chamberlain, K. M Scholfield, E.    Remaut, R. W. F. Le Page and J. M. Wells. Mucosal delivery of murine    interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus    lactis coexpressing antigen and cytokine. Infect. Immun., 1998;    66:3183-9.-   18. Medaglini D., G. Pozzi, T. P. King and V. A. Fischetti. Mucosal    and systemic immune responses to a recombinant protein expressed on    the surface of the oral commensal bacterium Streptococcus gordonii    after oral colonization. Proc. Natl. Acad. Sci. USA, 1995;    92:6868-72 McCracken V. J. and Gaskins H. R, ‘Probiotics a critical    review’, Horizon Scientific Press, UK 1999, p. 278.-   19. Marson, A., Kretschmer, K., Frampton, G. M., Jacobsen, E. S.,    Polansky, J. K., MacIsaac, K. D., Levine, S. S., Fraenkel, E., von    Boehmer, H and Young, R. A. Foxp3 occupancy and regulation of key    target genes during T-cell stimulation. Letters to Nature, 2007

The invention claimed is:
 1. An isolated strain of Bifidobacteriumdeposited at NCIMB with accession number
 41387. 2. The Bifidobacteriumstrain as claimed in claim 1 in the form of viable cells.
 3. TheBifidobacterium strain as claimed in claim 1 in the form of non-viablecells.
 4. A formulation comprising an isolated strain of Bifidobacteriumdeposited at NCIMB with accession number
 41387. 5. The formulation asclaimed in claim 4 which further comprises a probiotic material.
 6. Theformulation as claimed in claim 4 which further comprises a prebioticmaterial.
 7. The formulation as claimed in claim 4 further comprising aningestable carrier.
 8. The formulation as claimed in claim 7 wherein theingestable carrier is a pharmaceutically acceptable carrier.
 9. Theformulation as claimed in claim 7 wherein the ingestable carrier is afood product.
 10. The formulation as claimed in claim 1 which furthercomprises a protein and/or peptide, a lipid, a carbohydrate, a vitamin,mineral and/or trace element.
 11. The formulation as claimed in claim 4wherein the Bifidobacterium strain is present in an amount of more than10⁶ cfu per gram of the formulation.
 12. The formulation as claimed inclaim 4 which further comprises an adjuvant.
 13. The formulation asclaimed in claim 4 which further comprises a bacterial component. 14.The formulation as claimed in claim 4 which further comprises a drugentity.
 15. The formulation as claimed in claim 4 which furthercomprises a biological compound.
 16. A food stuff comprising an isolatedstrain of Bifidobacterium deposited at NCIMB with accession number41387.
 17. A medicament comprising an isolated strain of Bifidobacteriumdeposited at NCIMB with accession number
 41387. 18. An anti-inflammatorybiotherapeutic agent comprising an isolated strain of Bifidobacterium sdeposited at NCIMB with accession number
 41387. 19. An anti-inflammatorybiotherapeutic agent for reducing the levels of pro-inflammatorycytokines comprising a Bifidobacterium strain as claimed in claim
 1. 20.The formulation as claimed in claim 8 wherein the pharmaceuticallyacceptable carrier is in the form of a capsule, tablet or powder. 21.The formulation as claimed in claim 9 wherein the food product isselected from the group consisting of: acidified milk, yogurt, frozenyogurt, milk powder, milk concentrate, cheese spreads, dressings andbeverages.
 22. The formulation as claimed in claim 10 wherein theprotein and/or peptides are rich in glutamine/glutamate.